Project Summary Sequencing of genetic and epigenetic variants associated with disease states has revealed that ATP- dependent chromatin remodelers are among the most frequently disrupted genes in a number of diseases. In mammals, BAF (SWI/SNF), PBAF, and GBAF complexes are highly conserved ATP-dependent chromatin remodelers that generate accessibility for DNA-templated processes. Although these complexes have well- documented roles in many contexts, the mechanisms by which BAF-family complexes are regulated by specific signals remains poorly understood. Additionally, accessible sites across the genome respond with extreme heterogeneity to remodeling by these complexes, yet the basis of this heterogeneity, the physical origins of remodeling specificity, as well as the effects on transcription after initiation remain largely unknown. Improved understanding of these principles would provide powerful opportunities to manipulate gene expression in normal and disease states. To address this challenge, we will develop and combine new tools in epigenetics, chemical biology, and microscopy. We will make use of rapid technologies to manipulate BAF activity in living cells using cell-permeable molecules, and measure the outcomes using sensitive, unbiased approaches, including epigenomics and live-cell microscopy. We will use these tools to answer essential questions about ATP-dependent chromatin remodeling specificity, including: (1) How are BAF (SWI/SNF) complexes regulated via cellular signals? (2) Why do sites respond differently to chromatin remodeling? (3) How does chromatin remodeling influence transcription after initiation? Revealing the fundamental mechanisms used by these factors holds great promise to enable powerful intervention strategies for diverse human diseases.